恩诺沙星免疫胶体金快速检测试纸条的研制

发布时间：2019-04-24 21:49

【摘要】：本论文优化建立了恩诺沙星胶体金免疫层析分析方法,并有效用于动物源性食品、环境样品及动物代谢物中恩诺沙星的快速检测。本研究利用混合酸酐法合成恩诺沙星包被原,将恩诺沙星抗血清经protein A-sepharose4B进行纯化,得到抗恩诺沙星多克隆抗体。采用柠檬酸三钠还原法制备粒径为17nm胶体金,通过一系列优化,最终确定胶体金连接金标抗体最适pH为9,最佳标记1mg/mL恩诺沙星多克隆抗体用量为20μL。根据优化用量标记恩诺沙星多克隆抗体,制得胶体金标记抗体。以硝酸纤维素膜为固相载体,ENR-OVA包被原)和羊抗兔二抗分别作为检测线和控制线包被在硝酸纤维素膜上,金标记抗体喷涂在玻璃纤维垫上,制成快速检测恩诺沙星免疫层析试纸条。建立了胶体金免疫层析分析方法,检出限为5μg/L,在浓度为1000μg/L时,15种喹诺酮类药物及8种常用兽药对恩诺沙星免疫胶体金快速检测均无明显干扰,表明恩诺沙星胶体金免疫层析分析方法具有较高特异性。选取牛奶、猪肉、猪肝等9种动物源性产品、池塘水及猪尿作为检测样品,分别添加5,20,100μL的恩诺沙星标准品,经过简单的处理即可上样检测,牛奶、池塘水及猪尿样品检测限在5-25μg/L之间,其余动物源性产品检测限为50μg/kg,均低于最高残留限量。所建立的恩诺沙星免疫层析分析方法与酶联免疫分析方法的一致性较好,验证了方法的准确性。论文所开发的胶体金试纸条无需检测仪器,操作简单,快速,准确。实验结果稳定性高,可作为动物性食品中恩诺沙星残留现场大量筛选,及监测动物生长环境中恩诺杀星滥用的有效手段。
[Abstract]:In this paper, a gold immunochromatography method of enrofloxacin colloid was established and applied to the rapid detection of enrofloxacin in animal-derived food, environmental samples and animal metabolites. Enrofloxacin coated proto was synthesized by mixed anhydride method in this study. Enrofloxacin antiserum was purified by protein A-sepharose4B to obtain polyclonal antibody against enrofloxacin. The particle size of 17nm colloidal gold was prepared by tricarbonate reduction method. Through a series of optimization, the optimal pH and 1mg/mL enrofloxacin polyclonal antibody dosage were determined to be 9 and 20 渭 L for colloidal gold conjugated gold antibody and enrofloxacin polyclonal antibody respectively. Colloidal gold labeled antibody was prepared by labeling enrofloxacin polyclonal antibody according to the optimal dosage. Using nitrocellulose membrane as solid phase carrier and sheep anti-rabbit second antibody as detection line and control line respectively, the nitrocellulose membrane was coated on the nitrocellulose membrane, and the gold labeled antibody was sprayed on the glass fiber pad, respectively, using the ENR-OVA coating as the carrier and the sheep anti-rabbit second antibody as the test line and control line respectively. The rapid detection of enrofloxacin immunochromatographic test strip was made. A method of colloidal gold immunochromatography was established. The detection limit was 5 渭 g / L. When the concentration was 1 000 渭 g / L, 15 quinolones and 8 common veterinary drugs did not interfere with the rapid detection of enrofloxacin immunogold. The results showed that the gold immunochromatographic assay of enrofloxacin colloid was highly specific. Nine kinds of animal-derived products such as milk, pork, pig liver, pond water and pig urine were selected as detection samples, and enrofloxacin standard samples of 5,20100渭 L were added respectively. After simple treatment, samples can be detected, milk, etc. The detection limit of pond water and pig urine was 5-25 渭 g / L, and the detection limit of other animal-derived products was 50 渭 g / kg, lower than the maximum residue limit. The established method of enrofloxacin immunochromatographic assay was in good agreement with that of enzyme-linked immunosorbent assay, and the accuracy of the method was verified. The colloidal gold test strip developed in this paper does not require a detection instrument, and is simple, rapid and accurate. The results were stable and could be used as an effective method to screen the residues of enrofloxacin in animal food and monitor the abuse of enrofloxacin in animal growing environment.
【学位授予单位】：天津科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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